Cardiolipin Effects on Membrane Structure and Dynamics - Langmuir

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Cardiolipin Effects on Membrane Structure and Dynamics Joseph D. Unsay, Katia Cosentino, Yamunadevi Subburaj, and Ana J. García-Sáez*

Langmuir 2013.29:15878-15887. Downloaded from pubs.acs.org by KAROLINSKA INST on 01/28/19. For personal use only.

Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany, and German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ABSTRACT: Cardiolipin (CL) is a lipid with unique properties solely found in membranes generating electrochemical potential. It contains four acyl chains and tends to form nonlamellar structures, which are believed to play a key role in membrane structure and function. Indeed, CL alterations have been linked to disorders such as Barth syndrome and Parkinson’s disease. However, the molecular effects of CL on membrane organization remain poorly understood. Here, we investigated the structure and physical properties of CL-containing membranes using confocal microscopy, fluorescence correlation spectroscopy, and atomic force microscopy. We found that the fluidity of the lipid bilayer increased and its mechanical stability decreased with CL concentration, indicating that CL decreases the packing of the membrane. Although the presence of up to 20% CL gave rise to flat, stable bilayers, the inclusion of 5% CL promoted the formation of flowerlike domains that grew with time. Surprisingly, we often observed two membrane-piercing events in atomic force spectroscopy experiments with CL-containing membranes. Similar behavior was observed with a lipid mixture mimicking the mitochondrial outer membrane composition. This suggests that CL promotes the formation of membrane areas with apposed double bilayers or nonlamellar structures, similar to those proposed for mitochondrial contact sites. All together, we show that CL induces membrane alterations that support the role of CL in facilitating bilayer structure remodeling, deformation, and permeabilization.

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INTRODUCTION Cardiolipin (1,3-diphosphatidyl-sn-glycerol, CL) appears as a minor component of the plasma membrane in many types of bacteria (5−30% depending on the bacteria and stage in the life cycle) and in the mitochondria (5−10% in the outer membrane and about 20% in the inner membrane) and chloroplasts of eukaryotes.1,2 Nonetheless, it seems to play an important role in supporting many membrane-dependent processes and membrane protein functions: together with ATP, it acts as a cooperative allosteric inhibitor of cytochrome c oxidase,3 facilitates proton leakage in the mitochondrial intermembrane space,4 and crystallizes with photosynthetic centers from Rhodobacter sphaeroides5 and the cytochrome bc1 complex from yeast,6 implying a role in structure stabilization. CL is also important for maintaining membrane integrity in response to environmental stresses.2,7,8 CL-containing vesicles were shown to interact with cytochrome c and result in budding/folding events.9 In addition, CL has been proposed to participate in the regulation of programmed cell death. It allows specific targeting of truncated Bid (tBid) to the mitochondria10 and promotes its binding with interaction partners such as Bcl-xL.11 The activation of Bax, a pro-apoptotic pore-forming protein, by tBid in cooperation with CL results in the formation of supramolecular openings that are believed to be involved in the permeabilization of the outer mitochondrial membrane leading to the release of apoptotic factors.12,13 The relevance of CL in cell function is shown by its relationship to several health © 2013 American Chemical Society

disorders. Impairment of the ability to maintain proper CL levels in humans leads to diseases such as heart failure,14 diabetes,15 and Barth syndrome.16 Moreover, abnormal CL profiles in Tafazzin knock-down mice led to developmental cardiomiopathy and neonatal death.17−19 Despite its central role in cellular function, little is known about the molecular effects of CL on membrane structure and dynamics. Unlike other phospholipids, CL contains two phosphatidyl moieties joined by one glycerol molecule (Figure 1). This chemical structure results in a relatively small anionic headgroup and a large hydrophobic tail formed by four acyl chains. Because of the restricted movement resulting from its size, CL is also not able to form intra- or intermolecular hydrogen bonds that can stabilize the negative charge of the head. Therefore, the unshielded negative charge can easily interact with protons and cations in solution. The interaction reduces the effective size of the polar head, promoting the formation of nonlamellar structures.20,21 Early studies addressed the phase transition of CL from lamellar to inverted hexagonal phases in the presence of divalent cations, such as Ca2+, as a result of the interactions of the CL polar head with the cations.22,23 Received: July 16, 2013 Revised: August 20, 2013 Published: August 20, 2013 15878

dx.doi.org/10.1021/la402669z | Langmuir 2013, 29, 15878−15887

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EXPERIMENTAL SECTION

Preparation of Supported Lipid Bilayers. L-α-Phosphatidylcholine from egg (EPC), L-α-phosphatidylethanolamine (PE), L-αphosphatidylinositol from bovine liver (PI), phosphatidylserine from brain (PS), and cardiolipin (CL) from bovine heart were purchased from Avanti Polar Lipids (Alabaster, AL). CL was mixed with EPC in the following increasing molar proportions: 0, 5, 10, and 20%. We also used a lipid mixture that approximates the composition of the mitochondrial outer membrane (MitoMix), as in Lovell et al.13 and Bleicken et al.,32 with the following composition: EPC/PE/PI/PS/CL 48.5:27.2:9.9:10.0:4.4 mol %, respectively. A similar system was also prepared where CL was substituted by EPC. Planar supported bilayers were prepared as described in ref 33. Briefly, lipids were dissolved in chloroform at the desired molar concentration, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate salt (DiD-C18) or 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) (Molecular Probes, Eugene, OR) was added to the lipid mixtures at a 0.01 or 0.1% (mol/mol) concentration. The solvent was evaporated under nitrogen flux and then subjected to vacuum for at least 1 h. Lipid mixtures were rehydrated to a final concentration of 10 mg/mL in PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, and 137 mM NaCl, pH 7.2). Ten microliters of the mixture was diluted with SLB buffer (140 μL of 150 mM NaCl, 10 mM HEPES, pH 7.4). The suspension was then vortex mixed and bath sonicated until a clear suspension was obtained, indicating that small unilamellar vesicles were formed. The clear solution at 37 °C was placed in contact with the solid support. For the FCS experiments, the solid support was the glass surface of a coverslip. For confocal fluorescence imaging and AFM measurements, the solid support was freshly cleaved mica previously glued to a coverslip. CaCl2 was added to a final concentration of 3 mM and incubated at 37 °C for 2 min in the case of CL/EPC samples and 10 min for the MitoMix samples. We also prepared samples without calcium. The samples were rinsed several times with SLB buffer to remove CaCl2 and unfused vesicles and then allowed to equilibrate at room temperature before analysis. Confocal Microscope Imaging. SLBs were imaged using a commercial LSM 710 (Carl Zeiss, Jena, Germany) at 20 °C. The excitation light of a helium−neon laser at 633 nm or of an argon laser at 488 nm was reflected by a dichroic mirror (MBS 488/561/633) and focused through a Zeiss C-Apochromat 40×, NA = 1.2 waterimmersion objective onto the sample. The fluorescence emission was collected by the objective and directed by spectral beam guides to photomultiplier tube detectors. Fluorescence Correlation Spectroscopy. FCS experiments were conducted at 20 °C using the same microscope setup described above. Photon detection was carried out with the avalanche photodiodes of the ConfoCor3 module, which were also used for online correlation of the fluorescence intensity trace. Data analysis was performed with software written in MATLAB (MathWorks). The autocorrelation curves were fitted with a 2D diffusion model with elliptical Gaussian detection (eq 1) using a nonlinear least-squares fitting algorithm

Figure 1. Structure of the predominant species of (A) cardiolipin from bovine heart, 1,3-bis(1′,2′-dilinoleoyl-sn-glycero-3′-phospho)-sn-glycerol, and (B) phosphatidylcholine from egg, 1-palmitoyl-2-oleyl-snglycero-3-phosphocholine.

The effects of CL on membrane organization at physiologically relevant concentrations remain poorly understood.24,25 Diluting the lipid mixture with zwitterionic phospholipids such as phosphatidylcholine (PC) reduced the effect of calcium-CL polymorphism.26 Several groups studied26−30 the thermodynamic and structural properties of CL mixed with phosphatidylethanolamine (PE) and/or phosphatidylcholine (PC). These studies showed that the addition of CL to lipid mixtures might promote condensation (reduction in the area per lipid) in the presence of physiological salt concentrations. Binary mixtures of CL with PE or PC formed stable mixtures: on one hand, CL-PE systems formed membrane domains believed to be caused by the tendency of both lipids to form hexagonal phases;27−29 on the other hand, CL-PC systems completely mixed and formed only lamellar phases without any domains or structures.26,30 Here, we have examined how CL affects the lipid bilayer morphology and dynamics. We used supported lipid bilayers (SLB) made of EPC and CL at different concentrations and a lipid mixture mimicking the composition of the mitochondrial outer membrane (MitoMix). These systems were characterized by a combination of microscopy and surface analysis techniques31 including confocal microscopy, fluorescence correlation spectroscopy (FCS), and atomic force microscopy (AFM) using both imaging and force spectroscopy modes. Our results show that CL increases the fluidity and decreases the mechanical stability of the membrane, which is likely due to a decrease in lipid packing. Interestingly, at 5% CL we observed the formation of membrane domains, which grew with time. This correlated with the appearance of double membrane piercing events in force spectroscopy measurements on CLcontaining membranes. Taken together, these results indicate that CL promotes the formation of double bilayers and/or nonlamellar structures under physiologically relevant conditions, which support the role of CL in the deformation of biological membranes.

1⎛ τ ⎞ G(τ ) = ⎜1 + ⎟ N⎝ τD ⎠

−1/2

−1/2 ⎛ τ ⎞ ⎜1 + ⎟ τDS2 ⎠ ⎝

(1)

where G(τ) is the autocorrelation function, N is the number of fluorescent particles in the detection volume, τ is the lag time, S is the structure parameter (aspect ratio) of the Gaussian detection volume, and τD is the diffusion time (i.e., the average time a particle spends in the detection volume). The apparent diffusion coefficient, D, was calculated using

D=

ω0 2 4τD

(2)

where ω0 is the waist radius of the focal volume obtained from calibration measurements and τD is the diffusion time of the dye in the membrane derived from autocorrelation curves. 15879


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Figure 2. Confocal microscopy of bilayers with 0.01% DiD (red) or DiO (green). Bilayers prepared without calcium (A−C). (A) Fluid bilayer formed by EPC (inset: 50 × 50 μm2 close-up image of the indicated white square on the original image; some bright spots may be seen, and these are simply unfused vesicles adsorbed on the surface of the membrane). (B) 5% CL-EPC prepared without calcium produced a continuous phase (inset: 10 × 10 μm2 close-up image of the indicated white square). 10% CL-EPC (C) and 20% CL-EPC (results not shown) vesicles adsorbed only to the mica surface without undergoing rupture and bilayer formation (inset: 10 × 10 μm2 close up). (D−F) Bilayers prepared with calcium showed fluid bilayers (insets: 10 × 10 μm2 close-up image; dark regions are membrane defects). (G−I) Photobleaching experiments show that bilayers prepared in the presence of calcium, but not in the absence, are fluid. (G) CL-EPC bilayers prepared without calcium did not recover fluorescence after photobleaching, (H) whereas they did with calcium. (I) MitoMix also formed continuous fluid bilayers in the presence of calcium. Scale bars represent 50 μm in A−F and 100 μm in G−I. Atomic Force Microscopy. SLBs were imaged using a JPK NanoWizard II system (JPK Instruments, Berlin, Germany) mounted on an Axiovert 200 inverted microscope (Carl Zeiss). Intermittent contact (IC or tapping)-mode images were taken using V-shaped silicon nitride cantilevers with a typical spring constant of 0.08 N/m. The cantilever oscillation was tuned to a frequency of between 3 and 10 kHz, and the amplitude was set between 0.3 and 0.6 V. The amplitude was varied during the experiment to minimize the force of the tip on the bilayer. The scan rate was set to 0.7−1 Hz. The height, deflection, and phase-shift signals were collected simultaneously in both trace and retrace directions. The bilayer thickness was measured on the basis of the height profiles from the mica (membrane defects) to the membrane bulk. Force measurements were conducted as described in ref 34. Briefly, the calibration of sensitivity, resonance frequency, and effective spring constant (via the thermal noise method) of the cantilever was performed before each experiment. The total z-piezo displacement was set to 400 nm, and the indenting speed was set to 800 nm/s for the approach and 200 nm/s for the retraction. All experiments were carried out at different positions of the bilayer under the same conditions so that the effect of the speed of the breakthrough could be neglected. Force curves were processed using the accompanying JPK processing software. We applied a smoothing function, baseline correction, and tip−sample separation correction to the force curves to give accurate thickness and force measurements. We measured 300− 500 curves for each membrane system.

have a strong effect on CL polymorphism by inducing nonlamellar structures. To investigate its role in the formation and stability of CL-containing SLBs, we prepared CL-EPC bilayers in the absence or presence of calcium and imaged them using a laser scanning confocal microscope (Figure 2). In the absence of calcium, only pure EPC forms a continuous fluid phase (Figure 2A). CL-EPC bilayers (5%) form continuous phases with bright and dark regions (Figure 2B), but upon photobleaching, these regions did not recover their fluorescence (Figure 2G). CL-EPC (10%) and CL-EPC (20%) bilayers show vesicle adsorption on the mica but did not form continuous phases even after incubating overnight (Figure 2C). Washing the mica with buffer removes the adsorbed vesicles. Increasing the ionic strength of the buffer (through increased NaCl concentration) also does not produce bilayers, but washing preformed bilayers with EDTA-containing SLB buffer destroys the bilayers or left small membrane patches. In contrast, CL-EPC bilayers formed in the presence of calcium (which was subsequently removed by extensive washing) appeared as a single phase in confocal fluorescence microscopy (Figure 2D−F) that recovered after photobleaching at all of the CL concentrations studied (Figure 2H). These bilayers are stable for at least 4−8 h. These results indicate that trace amounts of calcium are necessary to prepare stable SLBs with natural concentrations of CL. To study the role of cardiolipin in a more physiological environment, we have used a lipid mixture mimicking the mitochondrial outer membrane lipid composition. Despite the increased complexity of this system compared to that of the EPC/CL bilayers, MitoMix bilayers show similar behaviors,

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RESULTS Effect of Calcium on the Formation and Stability of CL-Containing SLBs. Although SLB formation does not always require calcium, it induces vesicle rupture and adsorption on the mica support and hastens the process of bilayer formation.35,36 However, calcium has been shown to 15880


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Figure 3. Characterization of the CL effect on membrane fluidity by FCS. (A) Representative autocorrelation curves (fitted with a 2D diffusion model with elliptical gaussian detection) obtained for the different lipid compositions under study. (B) Apparent diffusion coefficient calculated for the corresponding membranes. The horizontal bars show the average values, and the errors correspond to the standard deviation. We have measured 75−80 FCS curves for the CL-EPC mixtures and 40 curves for the MitoMix membranes. A pairwise t test showed a significant difference under all conditions except those between the pairs: 10%/20% and 5%/20% CL-EPC.

Figure 4. AFM images of the bilayers surface. (A) Pure EPC. (B) 5% CL-EPC without structures at the edge of the defect. (C) 5% CL-EPC with structures at the edge of the defect. (D) 10% CL-EPC. (E) 20% CL-EPC. (F) MitoMix. The height profiles below each image correspond to the white line in the image. All images are 50 μm on each side. The diagonal lines and other patterns (that look like interference patterns) that appear in all images are artifacts of the AFM, which we cannot remove during image processing.

that measures the fluorescence fluctuations due to the diffusion of individual fluorophores in and out of the focal volume of the microscope. When the diffusion of lipophilic dyes introduced into the lipid bilayer is characterized, FCS can be used to probe the membrane fluidity.

forming a continuous homogeneous phase with only very few or no defects (Figure 2I). CL Increases the Fluidity of Phosphatidyl Choline Bilayers. To characterize the CL effects on membrane fluidity and packing, we used fluorescence correlation spectroscopy. FCS is a powerful technique with single-molecule sensitivity 15881


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Table 1. Summary of AFM Results for Cardiolipin-Containing Membranes force spectroscopya,b system

type of peak

0% CL-EPC 5% CL-EPC

A A B C

10% CL-EPC

20% CL-EPC

MitoMix

A B C A B C A B C

AFM imagingb

breakthrough force (nN) 5.4 1.4 6.8 0.9 3 7 1.0 1.5 3.5 8.8 1.1 4.7 7.6 4.2 7.4 4.3 5.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.4 0.8d 0.3d 0.1 6d 1d 0.7 0.1d 0.9d 0.8 0.6 1.6 1.3 0.6d 0.3d 0.4 0.4

bilayer thickness (nm)

c

roughness (nm)

2.5 ± 0.3 3.1 ± 0.6

2.9 ± 0.3 3.2 ± 0.2

5 ± 1e

4.9 ± 0.3f

5±2 6 ± 2e

6.0 ± 0.3

0.37 ± 0.06

5.8 ± 0.6 7.0 ± 1.1e

5.1 ± 0.9

0.33 ± 0.05

3.6 ± 0.1

4.5 ± 1.4

0.22 ± 0.07

0.28 ± 0.04 0.23 ± 0.05

4.7 ± 0.8e

Histograms from force and height distributions were fitted with a Gaussian function to elucidate the peak values. bErrors are given as the standard deviation of the distribution. cBilayer thickness was measured using two methods: height profiles from the AFM images and force spectroscopy. For AFM images, thickness was measured from mica (membrane defect) to the membrane bulk. For force spectroscopy, see Figure 6A. dTwo populations of breakthrough force resulted from the fitting of the force distribution. eFor curves with two peaks in force spectroscopy, the thickness from peaks B and C were added. fThickness of flowerlike domains from mica/bottom of the membrane. a

Figure 5. Growth of structures in 5% CL-EPC over time. Scale bars represent 20 μm.

the observations in simple CL-EPC bilayers and indicates that CL also increases the fluidity in membranes with more complex lipid compositions. Analysis of Membrane Topography by AFM Imaging. Atomic force microscopy scans the sample surface with a very sharp tip attached to a cantilever. It has an advantage over optical microscopy in that the spatial resolution is limited only by the tip sharpness and can easily go to a few nanometers. In addition, it provides a topographical image of the sample surface, which can reveal 3D features inaccessible by fluorescence microscopy. When we used AFM to image SLBs containing increasing amounts of CL, we found that pure EPC and bilayers made of 10% CL-EPC and 20% CL-EPC formed flat, homogeneous membranes with roughness in the 200−300 pm range (Figure 4 and Table 1). Interestingly, in the case of membranes made of 5% CL-EPC, we occasionally (about 33% of the time) but repeatedly observed the presence of domainlike structures at the edges of membrane defects. These domains were thicker than the surrounding membrane by 1.7 ± 0.2 nm. A time-lapse AFM experiment showed that these domains grew in size (within hours), merged, and eventually also formed a flat membrane (Figure 5). The AFM topological analysis of bilayers with MitoMix composition revealed a very flat surface with roughness and

We examined the dynamics of DiD in CL-EPC and MitoMix bilayers. Figure 3A shows the autocorrelation curves resulting from measuring the diffusion of DiD in the different membrane systems. As can be observed from the decay of the autocorrelation curves, the diffusion in CL-containing membranes is faster, which indicates a greater fluidity. By calibrating the size of the focal volume of the microscope with a soluble dye of known diffusion, we calculated the apparent diffusion coefficient of DiD for SLBs with different CL contents (Figure 3B). From the visual inspection of the curves, an increase in the diffusion coefficient values of DiD in the membrane was observed in the presence of CL compared to that in pure EPC membranes (4 ± 1 μm2/s). This increase was not linear and it reached a maximum value of 7 ± 2 μm2/s for concentrations of up to 10% CL, but no further changes were observed for a concentration of 20% CL (6 ± 2 μm2/s). The values obtained are typical for supported lipid bilayers in a liquid disordered state. In contrast, studies on the dynamics of MitoMix bilayers by FCS revealed a diffusion time of 6 ms and an apparent diffusion coefficient of 3.2 ± 0.8 μm2/s (Figure 3B), which are slower than those of the CL/EPC bilayers. When CL was substituted with EPC, the resulting diffusion coefficient value was even lower (2.5 ± 0.8 μm2/s, Figure 3B). This is in agreement with 15882


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Figure 6. Force spectroscopy curves of membranes. (A) Curves with single piercing events, peak A. (B) Curves with double piercing events, peaks B and C. (C) Percentage of curves with single (black) and double (red) piercing events for the different membrane compositions. Breakthrough force and thickness distribution of different membranes: (D, I) pure EPC, (E, J) 5% CL-EPC, (F, K) 10% CL-EPC, (G, L) 20% CL-EPC, and (H, M) MitoMix.

thickness values similar to those of membranes made of 5% CLEPC (Table 1 and Figure 4F). In this case, we did not observe domains. Effect of CL on the Mechanical Properties of the Membrane. In addition to imaging, AFM can also be used to probe the mechanical properties of the lipid bilayer when used in force spectroscopy mode. In these experiments, the AFM tip approaches the membrane surface in order to obtain force/ distance curves (Figure 6A,B). After contact, the tip continues to press the sample and the force increases linearly up to a certain value when there is a “jump” in the force. This is interpreted as the breakthrough force, which is the force needed to pierce the membrane. After measuring several hundred curves, we plotted a histogram of breakthrough forces.

When we used this method to test the effect of CL on the mechanical properties of the membrane, we obtained curves with single (Figure 6A) and double (Figure 6B) piercing events in CL-containing SLBs but not in pure EPC membranes. From here on, we refer to the peak on the curves with single piercing events as peak A. The first peak that appears in curves with double piercing events will be referred to as peak B, and the second peak will be referred to as peak C. We first consider the force curves with a single piercing event, peak A. CL decreased the breakthrough force of the bilayer in a concentration-dependent fashion: 5% CL-EPC clearly showed two distributions, one still close to the values of a pure EPC bilayer (6.8 ± 0.3 nN) and another close to much lower values (1.4 ± 0.8 nN). Increasing concentrations of CL gradually reduced the second distribution and decreased the 15883


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coupling of the membrane to the support, which can increase its breakthrough force. To verify the hypothesis that the double peaks reflect a different membrane organization, we plotted the thickness of the different membranes (Figure 6I−L). The thickness of the bilayer estimated from single piercing events increased with CL content from 2.5 ± 0.3 for pure EPC up to 5.8 ± 0.6 for 20% CL (Table 1). This is approximately the same value that we obtained from the sum of the thicknesses in the experiments with two piercing events (7.0 ± 1.1 nm for 20% CL) and roughly corresponds to double the thickness of a lipid bilayer in the liquid disordered state. In agreement with this, Table 1 also shows the roughness and thickness of the different membranes based on AFM images. Similar to CL-EPC bilayers, SLBs with MitoMix composition showed single and double piercing events in the force spectroscopy curves. Interestingly, in the case of single piercing events, the breakthrough force distribution was similar to that observed in 5% CL-EPC, with two main peaks (cf. Figure 6E,H), and the thickness values were also comparable (Table 1). We note that the central values for these two samples are different at 4.2 and 7.4 nN (cf. 1.4 and 6.8 nN in 5% CL-EPC). When two piercing events were present in the force curve, peak B overlapped the first population of peak A (4 nN), and peak C did not match the second population of peak A and showed values at 5 nN (Figure 6H). Moreover, when summing the thicknesses of peaks B and C, the resulting thickness value (4.7 nm) was greater compared to that for single piercing events (3.6 nm), but it did not double its value as in the case of 5% CL-EPC (Table 1).

force values (Figure 6D−F). These results suggest that CL decreases the mechanical stability of the lipid bilayer. The presence of double piercing events in force experiments has been quite controversial and not fully explained so far.37,38 However, the two peaks that we often obtained in CLcontaining membranes may reflect a special organization of the membrane, such as the presence of double bilayers or even nonlamellar structures (Figure 7). Indeed, we observed only

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DISCUSSION Cardiolipin, a unique lipid with four acyl chains, plays a key role in the normal functioning of the cell. To understand its effects on the organization of biological membranes, we have characterized the morphology and physical properties of CLEPC SLBs with a combination of microscopy-based techniques. In our studies, we found that incubation with calcium is necessary to prepare SLBs containing physiological amounts of CL. In agreement with our results, Richter and Brisson showed35 that negatively charged lipids did not form continuous bilayers on mica in the absence of calcium because of their inability to form stable interactions with the support. Calcium was also shown to induce the fusion of CL-containing vesicles,39 which is an important step in SLB preparation.35,40,41 This indicates the importance of electrostatic interactions in the adsorption and consequential rupture of CL-containing vesicles in forming stable, continuous bilayers. Our FCS measurements show that CL increases the bilayer fluidity compared to that of pure EPC bilayers, which is in contrast to previous studies.7,42 Although the surface roughness and interactions hinder the diffusion of lipids,43,44 distortions in the focal volume can lead to even more erroneous results.45,46 In our case, changing the support of the bilayer from mica (glued on a coverslip) to borosilicate glass reduced the signal/ noise ratio but led to similar results. Three reasons may explain the increased fluidity: (1) higher unsaturation introduced by the presence of CL (EPC has more than 50% unsaturated fatty acids whereas CL has 95% unsaturated fatty acids, based on product specifications from Avanti Polar Lipids, Inc.), which would make the bilayer more fluid;47,48 (2) concentration-dependent interactions between CL and EPC; and (3) the effect of calcium ions on the lipid

Figure 7. Models proposed for CL-EPC membrane polymorphisms. (A) 5% CL-EPC system with higher structures at the edge induced and stabilized by CL. (B) 20% CL-EPC as a double bilayer with a water layer in between. (C) 20% CL-EPC as a double bilayer with interacting layers induced by CL, which can span both bilayers. (D) 20% CL-EPC containing inverted micelles induced by CL interaction with calcium.

double piercing events in CL-containing membranes. Moreover, these events did not change with time, after changing the AFM tip or the researcher performing the experiments, supporting the fact that these observations were not an artifact. The breakthrough force for peaks B and C showed wider distributions than did peak A. Peak B generally follows the distribution of peak A for the different CL-containing mebranes, but peak C is higher and closer to the distribution of pure EPC. The higher values for peak C may indicate the 15884


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packing and structure. Nichols-Smith et al.30 showed that CL and EPC interact strongly as monolayers and condense in the presence of a salt subphase, implying that the addition of CL may hinder the lateral diffusion of lipid particles. These interactions are CL-concentration-dependent: there are stable and strong interactions for 5% CL-PC monolayers, but for 10% CL-EPC and 20% CL-EPC, these interactions become weaker and less stable. However, Khalifat et al. showed49 that interactions of the CL headgroups do not necessarily affect the fluidity of the hydrophobic core. The competition of attractive headgroup interactions versus increased hydrophobic core fluidity could explain why we do not see dramatic changes in the fluidity but rather the slight increase in fluidity from 5% CL-EPC to 10% CL-EPC and consequently a plateau from 10% CL-EPC to 20% CL-EPC. Another reason attributes diffusion to area per lipid molecule, wherein a study by Javanainen et al. showed50 that lipids in systems with lower area per lipid molecule diffuse more slowly than lipids in a system with a higher lipid area per molecule. In the previous study of NicholsSmith et al.,30 they showed that the excess area for the 5% CLPC system is greater than the excess area for 10% CL-PC and 20% CL-PC (which were comparable) in a salt subphase. This implies that the area per lipid molecule in 5% CL-PC is smaller than that of 10% CL-PC and 20% CL-PC and would consequently have a lower diffusion coefficient. Lastly, it has been shown that interactions between CL and calcium lead to changes in lipid packing and structure, increasing the isotropic and flip-flop motion of lipids51 and decreasing the barrier properties of CL-PC vesicles.52 These changes may also increase the lateral diffusion of lipidic dyes in the membrane. In the AFM imaging experiments, we found that all of the bilayers were flat and homogeneous, except for those made from 5% CL-EPC, which often presented membrane defects surrounded by higher structures that grew in time. Interestingly, the sum of the thickness of these higher structures with the normal thickness of the 5% CL-EPC bilayers gave a value similar to the thickness found for the 10% CL-EPC and 20% CL-EPC bilayers. This suggests that the 5% CL-EPC membranes are initially similar to pure EPC membranes and evolve with time toward membrane organizations similar to those observed for higher CL concentrations. Considering that the transition temperatures of 1-palmitoyl-2-oleoyl-sn-glycero3-phosphatidylcholine (main component of EPC) is −20 °C, CL from bovine heart is 19 °C, and 40% CL-EPC is 4 °C,28 most likely we are far away from the thermotropic transition temperatures of our membrane systems. It was reported, though, that the presence of a solid support may change the thermotrophic phase-transition temperature of lipids;53 however, this effect becomes prominent only when SLBs are prepared below their transition temperatures. Cooling hysteresis of cardiolipin24 may also play a role in the presence of these higher structures around the membrane in which kinetically trapped phases may persist depending on the cooling rate of the samples. On the basis of these evidences, we hypothesize that these higher structures could be double apposed bilayers or even nonlamellar structures (Figure 7). It is important to note that the formation of double bilayers also implies the presence of highly curved membrane regions, which may contain nonlamellar organization of the lipids in the contact zones between both bilayers (Figure 7A,B). For 5% CL-EPC, cardiolipin may induce the formation of these structures at the edge of membrane defects because of the local lipid packing defects.

These structures may represent a more stable organization of the lipids because they grow with time until the formation of a flat topography. Indeed, the flat membranes observed in the case of 10% and 20% CL-EPC might present this special lipid organization from the beginning as a result of the increased concentration of CL in these membranes. Although so far there have not been reported double bilayer formations for CL-EPC bilayers in this range of concentrations, force spectroscopy curves showing the presence of two peaks support our hypothesis. In addition, CL interacting with calcium could also play a role in the stabilization of the two apposed bilayers. Upon vesicle rupture, CL, because of its molecular shape, can induce the formation of inverted micelles. Alternatively, the two CL phosphatidyl moieties can also span the two bilayers. This would stabilize the double bilayer or may even produce a hypothetical trilayer. Furthermore, because mica may induce interleaflet flip-flop with anionic lipids,41 the bilayer leaflet interacting with mica may have a greater concentration of CL and form inverted hexagonal phases on the lower layer. It could also be that all structural arrangements are present in the CL-containing membranes characterized in this study. Although we cannot distinguish between these hypotheses, our results indicate that CL promotes the formation of lipid arrangements beyond the canonical lipid bilayer, which are likely to play a role in the functional structure of biological membranes containing CL. Indeed, some studies in CL-deficient mitochondria show different morphologies lacking cristae-like structures.54 Single-vesicle studies also show that CL allows for the formation of membrane invaginations under acidic pH conditions.49 Despite the higher complexity of the MitoMix composition, we found strong similarities with the 5% CL-EPC bilayers, containing a comparable amount of cardiolipin. In both examples, the presence of CL increases the fluidity of the bilayer. In the case of single piercing events, both systems show two clear populations in the distribution of the breakthrough force values and similar values of bilayer thickness, indicating that the same kind of membrane organization is present in lipid mixtures mimicking the mitochondrial outer membrane. Furthermore, this clearly points to the important role of cardiolipin in defining the structure of the bilayer, which in turn maintains the biological functions of these bilayers in bacteria and cells.54 However, MitoMix bilayers present homogeneous surfaces in contrast to the higher structures observed for 5% CL-EPC (1.7 nm above the membrane). Furthermore, in the MitoMix double piercing event curves, the distribution of peak B does not overlap the second population of peak A and the sum of the height values of peaks B and C does not double the thickness values of the single piercing events. These differences compared to the 5% CL-EPC system might be due to the higher complexity in the MitoMix lipid composition. It contains several negatively charged lipids, such as PI and PS, in addition to CL, presenting one or two monounsaturated or polyunsaturated chains. Furthermore, the interaction of PE with CL has been reported to induce the formation of domains at temperatures below the main phase-transition point of the system.55 Studies on the lipid propensity to form lamellar/nonlamellar structures56,57 show that negatively charged lipids and unsaturated acyl chains favor the formation of hexagonal HII phases in the presence of positive charges.56,57 Hexagonal phases from membrane phospholipids have thickness values in 15885


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the range of 4 to 5 nm.58,59 In this context, our findings may suggest the preferential formation of hexagonal phases in the MitoMix systems over that of other lamellar/nonlamellar structures. Furthermore, we have observed that MitoMix bilayers are less fluid compared to pure EPC bilayers. The high level of unsaturated chains in the MitoMix composition, on one hand, should lower the main transition-temperature point of this system and prevent the formation of domains at room temperature. On the other hand, it should increase the membrane fluidity47 compared to that of EPC bilayers, which, however, was not confirmed by our FCS measurements. It is possible that the decrease in the membrane fluidity observed in the MitoMix system cannot be attributed exclusively to the CL as suggested in refs 7 and 42 but might be due to repulsive electrostatic interactions between the different negatively charged lipids.60 Indeed, we have not observed any decrease in the membrane fluidity of the other model systems containing CL. In addition, our observation that MitoMix systems without CL exhibit a further decrease in the membrane fluidity supports this hypothesis.

REFERENCES

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CONCLUSIONS We have analyzed the impact of CL on the structure and dynamics of model lipid bilayers. We could prepare stable SLBs with concentrations ranging between 5 and 20% that looked homogeneous except in the case of 5% CL, which often presented flowerlike thicker lipid domains. We found that the fluidity of pure EPC membranes increased with the CL concentration. Interestingly, CL also increased the fluidity of membranes mimicking the mitochondrial outer membrane composition when compared to the same lipid mixture in the absence of CL. In all cases, CL decreased the force required to pierce the membrane, indicating a decrease in the mechanical stability of the lipid bilayer. Interestingly, we observed double membrane piercing events only in CL-containing membranes, which agreed in thickness with the formation of apposed double bilayers or other nonlamellar structures. Altogether, our results support the role of CL in the induction of membrane alterations involved in the remodeling, deformation, and permeabilization of biological membranes enriched in this lipid. The importance of such membrane alterations relates to the membranes’ function and ultimately the organisms’ survival; its physiological relevance is shown by the fact that CLdeficient mitochondria are linked to serious human diseases.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 0049 6221 5451234. Fax: 0049 6551 5451482. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Max Planck Society, the German Cancer Research Center, the Bundesministerium für Bildung und Forschung (grant N.0312040) and the DAAD (K.C.). We also thank Eduard Hermann for his help in analyzing AFM force spectroscopy curves, Dr. Jonas Ries for developing the FCS analysis program in MatLab, and Dr. Stephanie Bleicken for her valuable discussions with the authors. 15886


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Cardiolipin Effects on Membrane Structure and Dynamics - Langmuir

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